DESCRIPTION: (Verbatim from the Applicant's Abstract) The overall goal, regarding the structural parameters of nitric oxide synthase (NOS) that determine function, has not changed from the initial project period. The scope of this research project has developed significantly to include all three isoforms of NOS (neuronal NOS (NOS-1: nNOS), inducible NOS (NOS-2; iNOS), and endothelial NOS (NOS-2; eNOS)), each of which utilizes reducing equivalents from NADPH to form L-citrulline and NO through two successive oxygenation steps. Although the basic chemical mechanisms of the isoforms are similar, their extent of coupling of reducing equivalents to the production of oxygenated products, overall reaction rate, and regulation differ significantly. These differences are germane to the biological roles of these enzymes in neuronal signaling, control of immune responses to bacterial insult, and regulation of vasodilatation. It is important to determine their structural properties of the individual isoforms in order to develop methods for differentially regulating the activities of these enzymes through therapeutic intervention. It is hypothesized that, despite their similarities in requiring the same prosthetic groups and cofactors (FAD, FMN, Fe-protoporphyrin IX, and tetrahydrobioptein) to catalyze the same enzymatic reaction, sequence and structural differences have evolved in NOS isoforms to accommodate their individual functions. The specific aims are organized according to the two major protein domains shared by all three isoforms: 1) Heme-binding (Oxygenase) and 2) Flavin-binding (Reductase) domains. Specific Aim 1: Demonstrate the structural/functional significance of the dimerization of the oxygenase (heme-binding) domains of all three isoforms of NOS, determine the role of the metal center, identified by crystallography and biochemical studies as ZnS4 by the PI and collaborators, and identify protein-protein interaction sites important in regulating NOS function. Methods include site-directed/deletion mutagenesis, chemical cross-linking, and development of novel NOS constructs for crystallography and identification of interacting cellular components. Specific Aim 2: Ascertain the structural determinants of the function of the flavin-binding domain of the three NOS isoforms in controlling electron flow within this domain and between the flavoprotein and oxygenase domains using site-directed and deletion mutagenesis, cross-linking experiments, and fluoresceinated peptides to measure intra- and intermolecular protein-protein interactions.